Effects And Regulatory Mechanisms Of FTO On Adipogenesis Of Porcine Preadipocytes

The fat deposition of pigs is one of the main factors that affect the economic characteristics of pigs,which can directly affect the production efficiency,pork quality,reproductive performance and disease resistance of pigs,thereby affecting the economic benefits of pig breeding.Therefore,understanding of the fat deposition process and its regulatory mechanism is of great significance for improving the reasonable deposition of porcine fat and promoting the healthy development of the pig industry.The differentiation process of porcine adipocytes determines the body fat deposition of pigs,and adipogenesis is affected by many factors.Studies showed that epigenetic modifications play an important role in the regulation of adipocyte differentiation.N6-methyladenosine(N6-methyladenosine,m6A)is the most widely distributed and abundant methylation modification on eukaryotic mRNA.m6A can dynamically and reversibly regulate RNA metabolism and processing,which has become a hotspot in the research of epigenetics in recent years.FTO is the first identified m6A demethylase,and it is also the first discovered obesity gene,which is strongly associated with adipocyte differentiation.mRNA m6A methylation plays a significant role in many important biological processes such as development,cell differentiation,and carcinogenesis.Our previous study found that the content of subcutaneous fat and intramuscular fat of Jinhua pigs were significantly higher than those of Landrace pigs.While the mRNA m6A level in the adipose tissue of Jinhua pigs was significantly lower than that of Landrace pigs,suggesting that m6A level is negatively correlated with pig fat deposition.However,the effect of m6A modification on porcine adipogenesis and its regulatory mechanism is still unclear.Therefore,this study explored the effect of FTO and its m6A demethylase activity on the adipogenesis of porcine preadipocytes.From the aspects of signal pathway,mitotic clonal expansion and autophagy,we investigated the molecular mechanism of m6A in regulating porcine adipocyte differentiation.Base on the above studies,we discovered that epigallocatechin gallate(EGCG)regulates adipogenesis by targeting FTO-m6A pathway.The main results are as follows:1.Effect of FTO on adipogenesis of porcine preadipocytesWe found that knockdown FTO significantly inhibited adipogenesis,cellular triglyceride accumulation,and gene expression of adipogenic marker genes PPARy,C/EBPa and FABP4 in porcine adipocytes.Overexpression of FTO promoted porcine adipocytes differentiation,cell triglyceride accumulation and expression of adipogenic marker genes.By constructing FTO wild-type(FTO-WT)and catalytic mutant(FTO-MUT)plasmids,we found that overexpression of FTO-WT,but not FTO-MUT,promoted porcine adipogenesis,indicating that the effect of FTO on porcine adipocyte differentiation depends on its m6A demethylase activity.2.The mechanism of FTO in regulating adipogenesis of porcine preadipocytes2.1 FTO regulates the adipogenesis through JAK2-STAT3-C/EBP? pathwayWe found that FTO deficiency suppressed JAK2 expression,STAT3 phosphorylation transcription of C/EBP? in porcine preadipocytes,suggesting that FTO may regulate porcine adipogenesis through JAK2-STAT3-C/EBP? signaling pathway.Using meRIP-qPCR analysis,we found that knockdown FTO increased m6A level of JAK2 mRNA.Using mutagenesis technique and dual-luciferase assay,we found that knockdown of FTO could not reduce the protein expression of JAK2 when its m6A site was mutated,suggesting that FTO regulates its protein expression through m6A ruodification.To further explore the mechanism of m6A in regulating JAK2 expression,we used RIP-qPCR analysis and found that m6A binding protein YTHDF2 can bind to JAK2 mRNA.By the mRNA stability analysis,we found that knockdown of YTHDF2 improved mRNA stability of JAK2.Collectively,these results indicate that FTO regulates JAK2-STAT3-C/EBP? signaling pathway through m6A methylation,thereby promoting adipocyte differentiation.2.2 FTO regulates the adipogenesis through mitotic clonal expansionThe FTO inhibitor MA2 was used to treat porcine preadipocytes at different stages of differentiation,and we found that MA2 mainly inhibited adipogenesis in the early stage of differentiation(0-2 days).Using flow cytometry analysis,we found that knockdown of FTO upregulated the number of cells in G1 and S phases,reduced the number of cells in G2 phase.Furthermore,we demonstrated that FTO knockdown markedly decreased the expression of CCNA2 and CDK2,crucial cell cycle regulators,leading to delayed entry of cells into G2 phase.Moreover,the m6A levels of CCNA2 and CDK2 mRNA were significantly upregulated following FTO knockdown.We explored the mechanism of m6A in regulating CCNA2 and CDK2 expression and found that YTHDF2 recognized and decayed methylated mRNAs of CCNA2 and CDK2.These data reveal that FTO knockdown decreases protein expression of CCNA2 and CDK2,leading to inhibited cell cycle progression,thereby suppressing adipogenesis.2.3 FTO regulates the adipogenesis through autophagyThrough RNA sequencing,we found that knockdown FTO significantly reduced the gene expression of key autophagy genes ATG5 and ATG7 in porcine preadipocytes,suggesting that FTO regulates porcine adipocyte differentiation through autophagy pathway.Using immunofluorescence staining and transmission electron microscope observation,we showed that overexpression of FTO increased autophagy flux and the number of autophagosomes,and promoted adipocytes differentiation.While knockdown of FTO inhibited autophagy and adipogenesis,suggesting that FTO promotes adipocyte differentiation through autophagy pathway.Furthermore,we demonstrated that knockdown of FTO increased the m6A levels of ATG5 and ATG7 mRNA.Using mutagenesis technique and dual-luciferase assay,we found that knockdown of FTO could not reduce the protein expression of ATG5 and ATG7 when their m6A sites were mutated,suggesting that FTO regulates their protein expression through m6A modification.Further study identified that ATG5 and ATG7 were the targets of YTHDF2.Knockdown of YTHDF2 increased mRNA stability of ATG5 and ATG7.Together,these findings unveil that FTO targets and regulates the protein expression of ATG5 and ATG7 through m6A modification,activating autophagy,thereby promoting adipocyte differentiation.3.The mechanism of EGCG in targeting FTO to regulate adipocyte differentiationEGCG is the main component of green tea polyphenols.We treated preadipocytes with different concentrations(0,50,100,200 ?M)of EGCG and found that EGCG inhibited adipocyte differentiation in a dose-dependent manner.By detecting the expression of mitotic clonal expansion-related genes,we proved that EGCG reduced the protein and gene expression of CCNA2 and CDK2.Further studies found that EGCG reduced the protein expression of FTO and increased m6A levels of CCNA2 and CDK2 mRNA,suggesting that EGCG regulates the protein expression of CCNA2 and CDK2 through the FTO-m6Apathway.Moreover,we found that EGCG increased the gene and protein expression of YTHDF2.Overexpression of YTHDF2 reduced mRNA stability of CCNA2 and CDK2.The results indicate that EGCG blocks the mitotic clonal expansion process by targeting FTO,thereby inhibiting adipocyte differentiation.In summary,this study reveals that FTO positively regulates adipogenesis of porcine preadipocytes in an m6A demethylase activity-dependent manner.We elucidate the FTO promotes adipogenesis through JAK2-STAT3-C/EBP?,mitotic clonal expansion and autophagy pathway.We also find that the ECGG regulates adipocyte differentiation by targeting the FTO-m6A-YTHDF2 pathway.These works help us to better understand the biological process of porcine adipogenesis and its regulatory mechanism,which provides effective molecular targets for regulation of porcine fat deposition,and also lays a theoretical basis for the regulation of porcine fat deposition by nutritional means.